Proliferative and Anti-Inflammatory Effects of Resveratrol and Silymarin on Human Gingival Fibroblasts: A View to the Future.

Objectives
It has been demonstrated that polyphenol components such as silymarin and resveratrol have anti-inflammatory properties. Periodontitis is a chronic inflammatory disease that leads to the breakdown of dental supporting tissues and tooth loss. The purpose of this study was to investigate the anti-inflammatory effects of silymarin and resveratrol on lipopolysaccharide (LPS)-induced inflammatory response in human gingival fibroblasts (HGFs).


Materials and Methods
HGFs were treated with different concentrations of silymarin and/or resveratrol (25, 50, 100 and 200μg/ml). The effects of silymarin and resveratrol on cell viability and proliferation were assessed by MTT assay and cell cycle analysis, respectively. Also, HGFs were treated with silymarin and/or resveratrol and were stimulated with LPS. The levels of Interleukin-6 (IL-6) and IL-8 were assessed by enzyme-linked immunosorbent assay (ELISA).


Results
After treatment with silymarin, the viability of fibroblasts significantly increased, whereas treatment with resveratrol did not have any significant effect on cell viability. However, the combination of these flavonoids (50μg/ml silymarin and 100μg/ml resveratrol) significantly increased the viability of fibroblasts. Resveratrol significantly inhibited LPS-induced IL-6 and IL-8 secretion by HGFs, but silymarin did not show such a significant effect.


Conclusions
The findings of the present study demonstrated the anti-inflammatory effects of resveratrol and its combination with silymarin. Therefore, the combination of silymarin and resveratrol may be useful as a therapeutic agent for treatment of periodontal diseases.


INTRODUCTION
Biofilm formation has an important role in bacterial infections in humans, especially in periodontitis. Biofilm bacteria attack the host's defense system and increase its resistance to mechanical and chemical treatments through various mechanisms [1,2]. Lipopolysaccharide (LPS) is an endotoxin of gram-negative bacterial cell wall and plays an important role in maintaining the structural integrity of bacteria and activating the production of pro-inflammatory cytokines such as Interleukin-8 (IL-8), IL-6, tumor necrosis factoralpha (TNF-α) and cyclooxigenase-2 (Cox-2) in host cells [3][4][5][6]. Although full-mouth disinfection can decrease the serum levels of cytokines in patients with chronic periodontitis, some polyphenols also can be suggested in this regard [7]. Polyphenols are various compounds of herbal origin with antioxidant properties [5]. It has been stated that resveratrol, a polyphenol found in grapes and wine, has several antiinflammatory, anti-platelet and anti-carcinogenic properties [8]. Studies have shown that resveratrol suppresses inflammation by inhibiting the transcription function of nuclear factor-kappaB (NF-κB) [9,10]. Resveratrol suppresses the production of IL-1β and reactive oxygen species (ROS) and reduces the activity of Cox-2. It also suppresses and modulates Interferongamma (IFN-γ), monocyte chemoattractant protein-1 (MCP-1), TNF-α and IL-6 [10-14]. Silymarin, the polyphenolic fraction from the seeds of milk thistle (Silybum marianum, (L) Gaertn., family of Asteraceae), holds 70-80% flavonolignans and 20-30% non-identified oxidized polyphenol compounds [15]. This polyphenol has been used worldwide for many years as a complementary alternative medicine for treatment of hepatic diseases [16]. Silymarin has also been reported to stimulate DNA and protein synthesis [17], and may inhibit the related transcription factor, NF-κB, that regulates the expression of different genes involved in inflammation, cell protection and cancer [18][19][20]. It has been shown that silymarin can suppress inflammation and induce epithelialization in fullthickness wounds in rats [21]. Silymarin is effective in the prevention of skin damage by ultraviolet (UV) sunlight, and it also improves melasma lesions [22]. Human gingival fibroblasts (HGFs), as the most critical and accessible mammalian cell types in periodontal tissues, are one of the most convenient cell lines for in-vitro experiments [23]. Considering different observations regarding the protective and anti-inflammatory effects of silymarin and resveratrol, we investigated the separate and combined effects of these two drugs on the proliferation of HGFs, along with their anti-inflammatory effects. To study the combined effect of silymarin and resveratrol, the cells were treated with 50μg/ml silymarin and 100μg/ml resveratrol or with 100μg/ml silymarin and 200μg/ml resveratrol. Cell viability assay: Cell growth was examined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay (Sigma-Aldrich, Seelze, Germany). HGFs (5×10 3 /well) were seeded in 96-well plates and were treated with different concentrations of silymarin, resveratrol or both for 24 hours. Next, 20μl of MTT (5mg/ml) was added to each well, and the mixture was incubated for 3 hours. Subsequently, the medium was removed and 100μl of DMSO was added to each well to solubilize violet formazan crystals in the cells. The absorbance rate was determined by spectrophotometry at 570 nm wavelength.

Cell cycle analysis:
The cells were treated with resveratrol or silymarin at a 100μg/ml concentration for 24 hours. After the treatment, the cells were trypsinized and centrifuged at 1200 rpm for 5 minutes. The cells were washed in phosphate buffered saline (PBS, GIBCO Life Technologies, Paisley, UK), fixed with ice-cold 70% ethanol, and stored at -20°C for 24 hours. Next, the cells were centrifuged (5 minutes at 1200 rpm) and the pellets were resuspended in a solution of PBS containing 5µg/ml propidium iodide and 0.1mg/ml Ribonuclease (RNase) and were incubated for 30 minutes at room temperature. Finally, cellular DNA content was analyzed by flow cytometry, and the percentage of the cells in July 2017; Vol.14, No. 4 www.jdt.tums.ac.ir 205 Evaluation of the distribution of the groups was as follows: First, we analyzed the skewness and kurtosis of the groups by the "descriptive tool" in "descriptive statistics" software. One-way ANOVA was used to analyze the differences between the groups, followed by post-hoc Tukey's and Dunnett's tests. P-values lower than 0.05 were considered significant.

RESULTS
Cell viability assay: www.jdt.tums.ac.ir July 2017; Vol.14, No. 4 However, the survival rate of HGFs did not change significantly after resveratrol treatment. In order to determine the best combination of resveratrol and silymarin for cell survival, separate examinations were conducted based on the previously obtained results.
The results of treatment with three combinations of resveratrol and silymarin showed that cell viability The expression of IL-6 and IL-8 significantly increased in the presence of LPS compared to control samples (P=0.0013 and 0.0048, respectively, Fig. 3A). Silymarin at the concentrations of 50 and 100μg/ml had no significant effect on the IL-6 level in the supernatant when compared with LPS, while resveratrol at the concentrations of 100 and 200μg/ml significantly reduced the secretion of IL-6 in the supernatant (P=0.0213 and 0.0143, respectively). The two selected combinations of silymarin and resveratrol (50μg/ml silymarin with 100μg/ml resveratrol and 100μg/ml silymarin with 200μg/ml resveratrol) also showed a significant reduction of IL-6 level in the supernatant, which was more significant with regards to the former combination when compared with LPS (P=0.011 and 0.026, respectively, Fig. 3B, left). In addition, silymarin showed no significant effects on IL-8 levels at 50 and 100μg/ml concentrations, whereas resveratrol significantly decreased IL-8 levels at 100 and 200μg/ml concentrations (P=0.0208 and 0.0149, respectively). Two different combinations (50μg/ml silymarin with 100μg/ml resveratrol and 100μg/ml silymarin with 200μg/ml resveratrol) showed significant inhibitory effects on IL-8 secretion, which were more prominent with regards to the former combination when compared with LPS (P=0.0048 and 0.006, respectively, Fig. 3B, right).

DISCUSSION
Periodontal disease is one of the most common chronic inflammatory diseases. Periodontitis is a multifactorial microbial disease with a destructive inflammatory course. Bacterial factors and host response have key roles in the initiation and progression of almost all types of periodontal diseases. Despite the relationship between periodontal diseases and specific bacterial pathogens, studies in the recent two decades have indicated that most tissue injuries are due to the host response to infection and are not directly correlated with infectious factors. The quality of host response to stimuli determines the development or improvement of the disease [24]. Focus on the host response led to the development of Host Modulatory Therapy (HMT). The goal of this treatment is to balance the host's response and protective mediators according to a healthy condition [25]. Naturally occurring flavonoids have been established as anti-inflammatory factors [1][2][3][4][5]. Hence, the aim of this study was to investigate the anti-inflammatory effects of silymarin and resveratrol on LPS-induced HGFs in vitro. In order to find the most effective concentrations of silymarin and resveratrol, separate experiments were conducted by selecting different concentrations of each agent. Three different combinations of these concentrations were also selected, according to the previous results. Silymarin showed a significant effect on cell viability at the selected concentrations, which was more prominent at 50μg/ml concentration. In addition, the combination of low concentrations of the agents demonstrated the most noticeable positive effect on cell viability. The results indicated a proliferative effect for silymarin, which is reflected in its combination with resveratrol. This is consistent with previous studies showing that silymarin effectively stimulates DNA and protein replication in culture media [18], and increases survival and growth rate of stem cells in the clinical setting [26]. To investigate the effects of resveratrol and silymarin on various cell cycle phases of HGFs, a cell cycle study was conducted. Silymarin had no significant effect on various phases of the cell cycle. These results were in accordance with previous findings related to the inhibitory impacts of silymarin on T-cell proliferation [27]. After inducing the cells with LPS, the cell cycle histogram changed dramatically, and the sub-G1 phase population increased, which could be attributed to the production of oxygen free radicals [28]  . TNF-α, which has a key role in increasing matrix metallopeptidase-9 (MMP-9), IL-6, iNOS and IL-1β in fibroblasts, is significantly inhibited by resveratrol [15]. The present study confirms the findings of the aforementioned studies and introduces a highly effective anti-inflammatory role of resveratrol in combination with silymarin on LPS-induced fibroblasts. Nevertheless, further investigations would be beneficial for clarifying the principal mechanisms of this effect.

CONCLUSION
The results of the present study advocate the antiinflammatory effect of resveratrol and the combination of resveratrol and silymarin, which was proved by decreased secretion of IL-6 and IL-8 by LPS-induced HGFs, as one of the main cell lines involved in gingival inflammatory processes and periodontal diseases. These findings may suggest resveratrol as a natural compound for prevention and treatment of inflammations such as periodontitis. These results may propose that the combination of resveratrol and silymarin has strong anti-inflammatory effects on LPS-induced HGFs. Nevertheless, clarification of the fundamental mechanism of these effects may lead to the development of novel therapeutic approaches for the treatment of periodontitis.